CD4+T helper cells are not a homogeneous population but can be divided on the basis of cytokine secretion into at least two subsets termed T helper type 1 (Th1) and T helper type 2 (Th2) (see e.g., Mosmann, T. R. et al. (1986) J. Immunol. 136:2348-2357; Paul, W. E. and Seder, R. A. (1994) Cell 76:241-251; Seder, R. A. and Paul, W. E. (1994) Ann. Rev. Immunol. 12:635-673). Th1 cells secrete interleukin-2 (IL-2) and interferon-xcex3 (IFN-xcex3) while Th2 cells produce interleukin4 (IL4), interleukin-5 (IL-5), interleukin-10 (IL-10) and interleukin- 13 (IL-13). Both subsets produce cytokines such as tumor necrosis factor (TNF) and granulocyte/macrophage-colony stimulating factor (GM-CSF). In addition to their different pattern of cytokine expression, Th1 and Th2 cells are thought to have differing functional activities. For example, Th1 cells are involved in inducing delayed type hypersensitivity responses, whereas Th2 cells are involved in providing efficient xe2x80x9chelpxe2x80x9d to B lymphocytes and stimulating production of IgG1 and IgE antibodies.
There is now abundant evidence that the ratio of Th1 to Th2 cells is highly relevant to the outcome of a wide array of immunologically-mediated clinical diseases including autoimmune, allergic and infectious diseases. For example, in experimental leishmania infections in mice, animals that are resistant to infection mount predominantly a Th1 response, whereas animals that are susceptible to progressive infection mount predominantly a Th2 response (Heinzel, F. P., et al. (1989) J. Exp. Med. 169:59-72; Locksley, R. M. and Scott, P. (1992) Immunoparasitology Today 1:A58-A6 1). In murine schistosomiasis, a Th1 to Th2 switch is observed coincident with the release of eggs into the tissues by female parasites and is associated with a worsening of the disease condition (Pearce, E. J., et al. (1991) J. Exp. Med. 173:159-166; Grzych, J-M.,et al. (1991) J. Immunol 141:1322-1327; Kullberg, M. C., et al. (1992) J. Immunol. 148:3264-3270). Many human diseases, including chronic infections (such as with human immunodeficiency virus (HIV) and tuberculosis) and certain metastatic carcinomas, also are characterized by a Th1 to Th2 switch (see e.g., Shearer, G. M. and Clerici, M. (1992) Prog. Chem. Immunol. 54:21-43; Clerici, M and Shearer, G. M. (1993) Immunology Today 14:107-111; Yamamura, M., et al. (1993) J Clin. Invest. 91:1005-1010; Pisa, P., et al. (1992) Proc. Natl. Acad. Sci. USA 89:7708-7712; Fauci, A. S. (1988) Science 239:617-623). Furthermore, certain autoimmune diseases have been shown to be associated with a predominant Th1 response. For example, patients with rheumatoid arthritis have predominantly Th1 cells in synovial tissue (Simon, A. K., et al. (1994) Proc. Natl. Acad. Sci. USA 91:8562-8566) and experimental autoimmune encephalomyelitis (EAE) can be induced by autoreactive Th1 cells (Kuchroo, V. K., et al. (1993) J. Immunol. 151:4371-4381).
The ability to alter or manipulate ratios of Th1 and Th2 subsets requires an understanding of the mechanisms by which the differentiation of CD4 T helper precursor cells (Thp), which secrete only IL-2, choose to become Th1 or Th2 effector cells. It is clear that the cytokines themselves are potent Th cell inducers and form an autoregulatory loop (see e.g., Paul, W. E. and Seder, R. A. (1994) Cell 76:241-251; Seder, R. A. and Paul, W. E. (1994) Ann. Rev. Immunol. 12:635-673). Thus, IL4 promotes the differentiation of Th2 cells while preventing the differentiation of precursors into Th1 cells, while IL-12 and IFN-xcex3 have the opposite effect. One possible means therefore to alter Th1:Th2 ratios is to increase or decrease the level of selected cytokines. Direct administration of cytokines or antibodies to cytokines has been shown to have an effect on certain diseases mediated by either Th1 or Th2 cells. For example, administration of recombinant IL-4 or antibodies to IL-12 ameliorate EAE, a Th1-driven autoimmune disease (see Racke; M. K. et al. (1994) J. Exp. Med. 180:1961-1966; and Leonard, J. P. et al. (1995) J. Exp. Med. 181:381-386), while anti-IL-4 antibodies cure the Th2-mediated parasitic disease, Leishmania major (Sadick, M. D. et al. (1990) J. Exp. Med. 171:115-127). However, as therapeutic options, systemic administration of cytokines or antibodies may have unwanted side effects and, accordingly, alternative approaches to manipulating Th1/Th2 subsets are still needed.
The molecular basis for the tissue-specific expression of IL-4 in Th2 cells, or any T cell cytokine, has remained elusive. One possibility is the presence of repressor proteins that selectively silence cytokines. Transcriptional silencing has been well documented for bacteria, yeast and mammalian genes. Examples include E. coli thermoregulation genes (Goransson, M. et al. (1990) Nature 344:682-685), yeast xcex12 mating type genes (Keleher, C. A. et al. (1988) Cell 53:927-936) and mammalian MHC class I and TcRxcex1 genes (Weisman, J. D. and Singer, D. S. (1991) Mol. Cell. Biol. 11:4228-4234; Winoto, A. and Baltimore, D. (1989) Cell 59:649-655). Indeed, early experiments involving injection of IL-2 genomic DNA into Xenopus oocytes suggested the existence of a repressor protein for IL-2 in resting versus activated T cell extracts (Mouzaki, A. et al. (1991) EMBO J. 10:1399-1406). These studies suggested that the absence of IL-2 production in resting T cells was due to proteins that silenced the transcription of IL-2 by interacting with negative elements in the IL-2 promoter.
A second possibility is the existence of Th selective transactivators. A family of four related transcription factors called Nuclear Factor of Activated T cells (NF-AT), plays a key role in the regulation of cytokine gene expression (see e.g., Emmel, E. A. et al. (1989) Science 246:1617-1620; Flanagan, W. M. et al. (1991) Nature 352:803-807; Jain, J. et al. (1993) Nature 365:352-355; McCaffrey, P. G. et at (1993) Science 262:750-754; Rao, A. (1994) Immunol. Today 15:274-281; Northrop, J. P. et al. (1994) Nature 369:497). However, NF-AT family members can bind to and transactivate the promoters of multiple cytokine genes including IL-2 and IL-4 (Rooney, J. et al. (1995) Immunity 2:545-553; Szabo, S. J. et al. (1993) Mol Cell. Biol. 13:4793-4805; Flanagan, W. M. et al. (1991) Nature 352:803-807; Northrop, J. P. et al. (1994) Nature 369:497). Thus, they are not likely to be responsible for directing Th1- or Th2-specific cytokine transcription. Most, if not all, NF-AT binding sites in cytokine promoter regulatory regions are accompanied by nearby sites that bind auxiliary transcription factors, usually members of the AP-1 family. It has been shown that NF-AT and AP-1 proteins bind coordinately and cooperatively and are required for full activity of the IL-2 and IL-4 promoters. Different AP-1 proteins, specifically c-Jun, c-Fos, Fra-1, Fra-2, Jun B and Jun D, have been shown to bind to these sites (Rao, A. et al. (1994) Immunol Today 15:274-281; Jain, J. et al. (1993) Nature 365:352-355; Boise, L. H. et al. (1993) Mol. Cell. Biol. 13:1911-1919; Rooney, J. et al. (1995) Immunity 2:545-553; Rooney, J. et al. (1995) Mol. Cell. Biol. 15:6299-6310). However, none of these AP-1 proteins is expressed in a Th1- or Th2-specific manner and there is no evidence for the differential recruitment of AP-1 family members to the IL-2 or IL-4 composite sites (Rooney, J. et al. (1995) Mol. Cell. Biol. 15:6299-6310). Thus, neither NF-AT proteins nor the AP-1 family members c-Jun, c-Fos, Fra-1, Fra-2, Jun B and Jun D can account for the tissue-specific transcription of IL-4 in Th2 cells.
This invention pertains to methods for regulating Th1 or Th2 subsets by modulating the activity of a transcription factor that regulates expression of a Th2-specific cytokine gene. As described further herein, it has now been discovered that the tissue-specific expression of IL-4 in Th2 cells is not due to a repressor protein but rather to a Th2-specific transactivator protein. The proto-oncogene c-maf has now been demonstrated to be responsible for the tissue-specific expression of the Th2-associated cytokine interleukin-4. Moreover, ectopic expression of c-maf in cells other than Th2 cells (e.g, Th1 cells. B cells and non-lymphoid cells) leads to activation of the IL-4 promoter and, under appropriate conditions, production of endogenous IL-4.
Accordingly, in one aspect the invention provides a method for modulating production of a T helper type 2 (Th2)-associated cytokine by a cell. The method involves contacting the cell with an agent that modulates the activity of a transcription factor that regulates expression of a Th2-associated cytokine gene such that production of the Th2-associated cytokine by a cell is modulated. In particular, the agents of the invention act intracellularly to modulate the activity of a transcription factor that regulates expression of a Th2-associated cytokine gene. Preferably, the transcription factor is a member of the maf family. Most preferably, the transcription factor is c-Maf. The Th2-associated cytokine modulated in the method is preferably interleukin-4. In one embodiment, production of the Th2-associated cytokine (e.g., IL-4) is stimulated, for example in a cell that does not normally express the cytokine (such as a Th1 cell or a B cell). A variety of agents can be used to stimulate cytokine production, including a nucleic acid molecule encoding a maf family protein that is introduced into and expressed in the cell and chemical agents that enhance the expression or activity of an endogenous maf family protein in the cell. In another embodiment, production of a Th2-associated cytokine by a cell (e.g., a Th2 cell) is inhibited. A variety of agents can be used to inhibit cytokine production, including antisense nucleic acid molecules that are complementary to a maf family gene, intracellular antibodies that bind maf family proteins (e.g., in the cell nucleus), inhibitory forms of maf family proteins (e.g., dominant negative forms) and chemical agents that inhibit the expression or activity of an endogenous maf family protein in the cell. Cytokine production by the cell can be modulated in vitro or in vivo. In one embodiment, a cell is contacted with a modulating agent in vitro and then is administered to a subject to thereby regulate the development of Th1 and/or Th2 subsets in the subject.
In another aspect, the invention provides methods for regulating the development of Th1 or Th2 subsets in a subject. In addition to the embodiment discussed above wherein ex vivo modified cells are administered to the subject, in another embodiment, these methods involve direct administration to the subject of an agent that modulates the activity of a transcription factor (e.g., a maf family member) that regulates expression of a Th2-associated cytokine gene (e.g., IL-4) such that development of Th1 or Th2 cells in the subject is modulated.
The methods of the invention can further involve the use of additional agents that modulate the activity of additional transcription factors that contribute to regulating the expression of Th1- or Th2-associated cytokines. Preferred additional agents are those which modulate the activity of a Nuclear Factor of Activated T cells (NF-AT) protein. Thus, in one embodiment, a stimulatory method of the invention can involve the use of a first agent that stimulates the expression and/or activity of a maf protein and a second agent that stimulates the expression and/or activity of an NF-AT protein. Similarly, an inhibitory method of the invention can involve the use of a first agent that inhibits the expression and/or activity of a maf protein and a second agent that inhibits the expression and/or activity of an NF-AT protein. Alternatively or additionally, the modulatory methods of the invention can involve the use of additional agents that modulate the activity of an AP-1 family protein.
The modulatory methods of the invention can be used to manipulate Th1:Th2 ratios in a variety of clinical situations. For example, inhibition of Th2 formation may be useful in allergic diseases, malignancies and infectious diseases whereas enhancement of Th2 formation may be useful in autoimmune diseases and organ transplantation.
Another aspect of the invention pertains to compositions that are useful for modulating the production of a Th2-associated cytokine by a cell and/or for modulating the development of Th1 or Th2 subsets in a subject. In one embodiment, these compositions include recombinant expression vectors that encode a maf family protein, wherein the maf-encoding sequences are operatively linked to regulatory sequences that direct expression of the maf family protein in a specific cell type, such as lymphoid cells (e.g., T cells or B cells) or hematopoietic stem cells. In another embodiment, these compositions include host cells, such as host lymphoid cells (e.g., host T cells or host B cells) or host hematopoietic stem cells, into which a recombinant expression vector encoding a maf family protein has been introduced.
Yet another aspect of the invention pertains to screening assays for identifying compounds that modulate the activity of a transcription factor that regulates expression of a Th2-associated cytokine gene. In one type of screening assay, an indicator cell which contains both 1) a recombinant expression vector encoding a transcription factor that regulates expression of a Th2-associated cytokine gene and 2) a vector comprising regulatory sequences of the Th2-associated cytokine gene operatively linked a reporter gene is used to identify compounds that modulate the expression and/or activity of the transcription factor. In another embodiment, a screening assay of the invention identifies proteins from Th2 cells that form a protein-protein interaction with a transcription factor (e.g., c-Maf) that regulates expression of a Th2-associated cytokine gene. In yet another embodiment, a screening assay of the invention identifies compounds that modulate the interaction of c-Maf with a maf response element (MARE) in the promoter of a Th2-associated cytokine gene (e.g., a MARE in the IL-4 promoter).